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International Food Research Journal 27(5): 963 - 970 (October 2020)Journal homepage: http://www.ifrj.upm.edu.my
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Abstract
Fruits are great sources of phytochemical compounds and multiple essential nutrients. Fruit peels can be exploited to reduce agro-waste as natural bioactive compound owning to the antimicrobial properties. The present work envisaged to explore the phytochemical compounds in aqueous and ethanol extracts of three fruit peels from pomegranate (Punica granatum), banana (Musa acuminata) and orange (Citrus reticulata). Additionally, it was aimed to ascertain the antibacterial activities of fruit peel extracts against six human bacterial pathogens; Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, and Staphylococcus aureus. Assessment of phytochemical proper-ties of pomegranate, banana, and orange peels revealed that these peels were rich in many bioactive compounds especially alkaloid, flavonoid, phlobatannins, saponins, terpenoids, glycosides, anthocyanosides, steroids, phenols, proteins, and carbohydrates. The antibiogram analysis was performed using four different antibiotics including cepharadin, ceftriaxone, pipemidic acid, and ofloxacin. Antimicrobial profiling indicated that the studied microorgan-isms were resistant to cepharadin and ceftriaxone, but susceptible to pipemidic acid and oflox-acin. S. aureus was found to be resistant against all applied antibiotics. On the other hand, ethanol extracts of pomegranate peels showed maximum inhibitory activities (29.66 ± 1.63 mm) against M. luteus, banana peels (27.00 ± 0.33 mm) against M. luteus, and orange peels (29.00 ± 0.57 mm) against K. pneumonia. Collectively, similar to the whole fruit, the fruit peels are carrier of crucial nutrients and can be exploited for the production of novel and natural therapeutics to substitute the synthetic drugs and leveraging the development of potent novel antibiotics.
Keywords
Article history
Received: 14 September 2019Received in revised form: 1 December 2019Accepted:16 December 2019
pomegranate, banana, orange, phytochemical, antibiogram, antibacterial
Introduction
The use of naturally occurring nutrients and organic foods are gaining interest in recent times. These constituents are usually recognised as bioactive phyto-chemicals. It has previously been reported that several synthetic drugs carry multiple toxicities and side effects (Luciano and Perazella, 2014; Cohen and Weinstein, 2018); therefore, development of novel therapeutics as well as antimicrobials especially against infectious diseases caused by multidrug-resistant pathogens are inevitable. Phytochemicals are loosely defined as polyphenols, flavonoids, isoflavonoids, anthocyani-dins, phytoestrogens, terpenoids, carotenoids, limo-noids, phytosterols, glucosinolates, and fibres (Pagliarulo et al., 2016). These phytochemicals may individually or synergistically facilitate to reduce the risk for a range of chronic and inflammatory disorders (Hossen et al., 2019), which include arterial sclerosis, disseminated multiple sclerosis, stroke, several types
of cancers, diabetes mellitus, allergy, asthma, arthritis, Crohn’s disease, Alzheimer’s disease, psoriasis, septic shock, Acquired Immunodeficiency Syndrome (AIDS), and neurodegeneration (Cseke et al., 2016). As a consequence, phytochemicals carry tremendous impact on the healthcare system and have potentials to prevent diseases and physiological disorders. Majority of plant-based foods such as whole grains, beans, fruits, vegetables, and herbs contain phytochemicals (Septembre-Malaterre et al., 2018). The utilization of plant extracts and phytochemicals is of great importance in therapeutics. During recent years, multiple studies have been conducted in several countries to prove such potencies (Islam et al., 2018). However, most of the plant peels are largely dumped as solid waste and not utilized for other applications. For example, 40% of the whole weight of banana is generated as waste material in banana-based products in manufacturing industries (Nagarajaiah and Prakash, 2011). The banana peel should and could be exploited
1Department of Zoology, University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan2Department of Zoology, Government College University, Lahore, Pakistan
3Department of Zoology, University of the Punjab, Lahore, Pakistan
1Gillani, S. F., 2*Ali, S., 2Tahir, H. M., 3Shakir, H. A. and 2Hassan, A.
Phytochemical analysis, antibacterial and antibiogram activities of fruits peels against human pathogenic bacteria
964 Gillani, S. F., et al./IFRJ 27(5) : 963 - 970
as a natural, eco-friendly, and economic supply of valu-able elements while solving the environmental issues. The present work focuses on the utilization of fruit peel to leverage waste management and antibiotic resistance which appears as an emerging challenge around the globe.
Materials and methods
Collection and preparation of dried plant material Fresh fruits i.e., pomegranates (Punica grana-tum), bananas (Musa acuminate), and oranges (Citrus reticulata) were collected from the fruit market of Azad Jammu and Kashmir, Pakistan. Fruits were thoroughly washed with running tap water and then with distilled water. The peels of each of fruits were separated and dried at room temperature (25 - 30°C) for 2 – 3 h. The dried peels were ground using a grinder. Fine powder was obtained and stored in polybags as described by Sharma et al. (2017) until further use.
Aqueous extraction For the preparation of aqueous extraction, 1 g of powder from each of fruit peels was added to 100 mL of distilled water individually in conical flasks and left for 1 day in a magnetic stirrer. Following this, the resulting aqueous extract was filtered through What-man filter paper I.
Ethanolic extraction For the preparation of ethanolic extract, powder of each peels was subjected to Soxhlet extrac-tion with 99% ethanol for 24 h. The mixture was evapo-rated to dryness in a rotary evaporator, and refrigerated. The condensed extracts were used for preliminary screening of phytochemicals as reported by Velumani (2016).
Phytochemical analysesDetection of alkaloids The preliminary phytochemical analysis of orange, banana, and pomegranate peels was performed on aqueous extract for the presence of alkaloids, flavo-noids, tannins, phlorotannins, anthraquinones, sapon-ins, terpenoids, anthocyanosides, steroids, glycosides, phenols, carbohydrates, and proteins by adopting the phytochemical procedure defined by Harborne (1998). Briefly, aqueous extract (1 mL) of each of the orange, banana, and pomegranate peel powder was added in a test tube with several drops of Wagner’s reagents. The presence of alkaloids was indicated by the forma-tion of reddish-brown precipitation.
Detection of flavonoids
The presence of flavonoids in the peels of orange, banana, and pomegranate was confirmed using 10% ammonium hydroxide in 1 mL of aqueous extract of the powdered peel of each of three fruits separately, and the formation of yellow colour confirmed their presence.
Detection of tannins The formation of brownish green or bluish-black colour after treating aqueous extract of peels with 0.1% FeCl2 indicated the presence of tannins in the extract.
Detection of phlorotannins The formation of reddish precipitation after boiling 1 mL of aqueous extract of peels with 1% HCl2 indicated the presence of phlorotannins in the extract.
Detection of anthraquinones The presence of anthraquinones was exam-ined by performing Borntrager’s test. Briefly, 1 mL of the aqueous extract of peels was hydrolysed with diluted H2SO4 and later removed with benzene. Then, 1 mL of diluted ammonia was added to the extract. Rose pink colour formation indicated the presence of anthraquinones in the extract.
Detection of saponins Foam test was used for the detection of sapon-ins. Briefly, 10 mL of sterile distilled water was added to 2.5 mL aqueous extract of peels in a test tube and was shaken thoroughly for 30 s. Following shaking, it was allowed to stand for 30 min. Formation of honey-comb froth / foam indicated the presence of saponins in the extract.
Detection of terpenoids Salkowski’s test was used to confirm the presence of terpenoids in the extract by mixing 1 mL of aqueous extract of peels in 0.5 mL of concentrated H2SO4 and chloroform. The red-brown colour in the interface formed indicated the presence of terpenoids in the extract.
Detection for glycosides The pink colour formed by mixing 2 mL of aqueous extract of peels with 3 mL of chloroform and 1 mL of 10% ammonium solution indicated the presence of glycosides in the extract.
Detection of anthocyanosides The formation of pale pink colour upon mixing 1 mL of aqueous extract of peels and 5 mL of diluted HCl indicated the presence of anthocyanosides
Gillani, S. F., et al./IFRJ 27(5) : 963 - 970 965
in the extract.
Detection of steroids The red-brown ring at the interface formed by adding 0.5 mL of aqueous extract of peels to 2 mL of chloroform and 1 mL of concentrated H2SO4 indicated the presence of steroids in the extract.
Detection of phenols The formation of green or blue colour by adding 1 mL of aqueous extract of peels to 2 mL of distilled water and a few drops of 10% ferric chloride indicated the presence of phenols in the extract.
Detection of proteins A total of 1 mL of aqueous extract of peels was boiled with 1 mL of 0.2% solution of ninhydrin. The formation of violet colour indicated the presence of amino acids and proteins in the extract.
Detection of carbohydrates By boiling the aqueous extract of peels with 1 mL of Benedict’s reagent, reddish-brown precipita-tion was formed which indicated the presence of carbo-hydrates in the extract.
Antibacterial activity by agar well diffusion method The antimicrobial activity of aqueous extract of peels was assessed by the agar well diffusion method (Rios et al., 1988). Nutrient agar (Oxoid, UK) and nutrient broth (Oxoid, UK) were used for bacterial cultivation. The bacteria were activated by inoculating a loopful in 25 mL of nutrient broth and incubated at 37°C on a rotary shaker for 24 h. The overnight culture was mixed with freshly prepared nutrient agar at 45°C and poured into sterile Petri dishes (pour-plate method). Inoculated Petri dishes were kept at room temperature in a laminar flow for solidification. Following this, in each plate, three wells of 5 mm diam-eter were made. Approximately 30 µL of each of the crude extracts of fruit peel powder and control solvent were placed in each prepared well, and plates were incubated at 37°C for 24 - 48 h. All solvents were also used as negative control. Microbial growth was determined by measur-ing the diameter of the zone of inhibition after 24 h in millimetre (mm). The diameter of the clear zones (if greater than 5 mm) around each well was measured with the help of the scale.
Antibiogram assay for antibiotic susceptibility test Antibiotic susceptibility of human pathogens was determined by Kirby Bauer disc diffusion test on Mueller-Hinton agar (Biemer, 1973) by using four
different antibiotics (cepharadin, ceftriaxone, pipem-idic acid, and ofloxacin). For this purpose, six human clinical bacterial pathogens were used including Escherichia coli, Bacillus subtilis, Klebsiella pneumo-niae, Micrococcus luteus, Pseudomonas aeruginosa, and Staphylococcus aureus. These pathogenic strains were acquired from Laboratory of Microbial Biotech-nology, Department of Zoology, Azad Jammu and Kashmir University, Muzaffarabad, Pakistan.
Statistical analysis Statistical analysis was performed using Graph-Pad Prism for Windows (version 5.03). To analyse the antibacterial activity of standard antibiot-ics and fruit peelts, one-way analysis of variance and Dunnett’s multiple comparison tests with a probability level of 5% as the minimal criterion of significance were used. Data are presented as means ± SEM.
Results and discussion
Preliminary screening of powdered peel of pomegran-ate, banana, and orange Preliminary phytochemical analysis showed that peel extracts of all three fruits were rich in phyto-chemicals in aqueous as well as in ethanol solutions. Different coloration in different reagents showed the presence of different phytocompounds in the plant extracts. In the present work, phytochemical analysis has revealed that fruit waste was a significant source of organic compounds including alkaloids, flavonoids, and glycoside with antibacterial activity against differ-ent pathogenic and drug-resistant bacteria by reducing membrane fluidity. A phenolic compound in the peel of the fruit may interact with enzymes responsible for the formation of the bacterial cell membranes (Miklasińska-Majdanik et al., 2018). The phlobatan-nins were absent in both aqueous and ethanol extracts of each of three fruit peels. It was revealed that pome-granate peel was rich in alkaloids, flavonoids, anthocy-anosides, tannins, phenols, and carbohydrates; where-as banana peel contained alkaloids, flavonoids, tannins, phenols, proteins, saponins, anthocyanosides, glycosides, terpenoids, and carbohydrates. Orange peel was rich in alkaloids, flavonoids, tannins, phenols, proteins, saponins, glycosides, steroids, and carbohy-drates. Many phytocompounds have been found in pomegranate peels such as gallagic acid, gallotannins, punicalins (García-Villalba et al., 2015), punicalagin, punicalins (Sun et al., 2017), esters of hexahydroxy-diphenic acid, and aldohexose or quinic acid (Clifford, 2004), glycosides (Singh et al., 2018), anthocyanidins (Akhtar et al., 2015), and flavonoids (Kumar and
966 Gillani, S. F., et al./IFRJ 27(5) : 963 - 970
Neeraj, 2018). The potential applications of banana peels rely on its chemical composition since complete-ly different fruit elements contain different inhibitors and antimicrobial elements. Unripen banana peels contain copper, zinc, sodium, potassium, calcium, phosphorus, and iron (Singh et al., 2016). Additional-ly, banana peel contains several nutrients such as dietary fibres, essential amino acids, proteins, and unsaturated fatty acids (González-Montelongo et al., 2010). Citrus fruits carry components of peculiar fragrance because of flavonoids and limonoids within the peel and these fruits are rich sources of antioxidants and flavonoids (Oikeh et al., 2016). The antioxidant / radical scavenging capabili-ty and reducing ability of various extracts of peels were investigated and results showed that ethanolic extract carried best values for yield i.e., total phenolic contents, total flavonoid contents, chelating, and inhib-itor activities (% DPPH scavenging activity). It had been demonstrated that solvents play an important role in the extraction of the plant constituents. Specifically, methyl alcohol and ethyl alcohol were extremely polar among the solvents used in the present work (Hegazy and Ibrahium, 2012).
Antibacterial activity of aqueous and ethanolic extracts of powdered peel of pomegranate, banana, and orange against six human clinical pathogenic bacteria Antibacterial activities of aqueous and ethanolic extracts against six human bacterial patho-gens were performed using agar well diffusion method as shown in Table 1. Antibacterial activities of ethanol-ic extract of pomegranate peel showed maximum zone of inhibition against M. luteus (29.66 ± 1.63 mm), whereas aqueous extract exhibited maximum
antibacterial activity against E. coli (24.33 ± 0.88 mm). Antibacterial activity of ethanolic extract of banana peels was highest against M. luteus (27.00 ± 0.33 mm), while aqueous extract exhibited maximum antibacteri-al activity against B. subtilis and M. luteus (23.00 ± 1.13 mm). Ethanolic extracts of orange peel had a max-imum zone of inhibition against K. pneumonia (29.00 ± 0.57 mm) and aqueous extract exhibited highest antibacterial activity against M. luteus (26.33 ± 0.33 mm). The results showed that the waste material of fruits (pomegranate, orange, and banana) contained high potency against human pathogenic bacteria. The results of the present work showed that bioactive compounds in fruit peels have a potent antibacterial effect. It has also been revealed from many previous studies that fruit peels carry highest antibacterial activity compared to other parts of fruit because of the presence of bioactive compounds. These activities were mainly attributed to flavonoids, phenolics, and tannins. Both aqueous and ethanolic extracts showed the antibacterial potential of peels of the fruits. However, ethanolic extract showed a signifi-cantly large inhibition zones as compared to aqueous solution because alcohol is a better solvent for active antimicrobial compounds (Sadeghian et al., 2011). As reported by antimicrobial principles, either polar or non-polar organic solvent medium are more favoura-ble for phytochemicals extraction (Ehiowemwenguan et al., 2014).
Antibiogram assay Antibiotic susceptibility of studied bacteria was determined by Kirby Bauer disc diffusion assay on Mueller-Hinton agar using antibiotics including cepharadin, pipemidic acid, ceftriaxone, and ofloxa-cin. The results showed that the B. subtilis showed
Table 1. Antibacterial activity of aqueous extract and ethanol extract of pomegranate, orange, and banana peel powder against human pathogenic bacteria.
Pathogen
Zone of inhibition in mm (mean ± SEM)
Pomegranate
(Punica granatum) peel
Banana
(Musa acuminate) peel
Orange
(Citrus reticulata) peel
Ethanolic
extract
Aqueous
extract
Ethanolic
extract
Aqueous
extract
Ethanolic
extract
Aqueous
extract
Bacillus subtilis 28.33 ± 1.68** 24.00 ± 0.60 23.33 ± 1.49 23.00 ± 0.67 27.66 ± 0.33 24.66 ± 0.33
Micrococcus luteus 29.66 ± 1.63** 24.00 ± 0.60 27.00 ± 0.33 23.00 ± 1.13 27.66 ± 0.57 26.33 ± 0.33
Pseudomonas aeruginosa 23.00 ± 0.94 22.00 ± 0.51 20.33 ± 1.23 20.33 ± 0.94 24.0 ± 0.57** 23.00 ± 0.57
Staphylococcus aureus 27.33 ± 1.98** 23.33 ± 0.99 21.33 ± 1.12 21.00 ± 0.84 24.33 ± 0.33 23.33 ± 0.33
Klebsiella pneumonia 28.33 ± 0.33 23.66 ± 0.33 25.00 ± 0.57 22.66 ± 0.33 29.0 ± 0.57** 25.33 ± 0.33
Escherichia coli 27.00 ± 0.57 24.33 ± 0.88 25.00 ± 0.57 22.00 ± 0.57 28.3 ± 0.66** 23.66 ± 0.33
Gillani, S. F., et al./IFRJ 27(5) : 963 - 970 967
resistance against cepharadin and ceftriaxone; and susceptibility against ofloxacin and pipemidic acid. K. pneumoniae appeared resistant against cepharadin and ceftriaxone; and susceptible against ofloxacin and pipemidic acid. On the other hand, M. luteus showed resistance against cepharadin and ceftriaxone; and susceptibility against ofloxacin and pipemidic acid. P. aeruginosa showed resistance against cepharadin and ceftriaxone; and susceptibility against ofloxacin and pipemidic acid. S. aureus showed resistance against cepharadin and ceftriaxone; and susceptibility against ofloxacin and pipemidic acid. E. coli showed resistance against cepharadin and ceftriaxone; and susceptibility against ofloxacin and pipemidic acid as shown in Figure 1. Antibiogram analysis against the six pathogenic strains are shown in Figure 1, Figure 2, and Table 2. The statistical analysis revealed that there was a significant difference in inhibition of the six tested human pathogens between peel extracts (ethanolic and aqueous) and standard available antibiotics (p < 0.05). This may be due to antibiotic resistance of pathogens
against antibiotics whereas fruit peels had bioactive compounds that have potential against pathogenic bacteria, thus can be further utilised as alternative for antibiotics. The phytochemical compounds play a vital role against different microorganisms (Singh et al., 2018) and are vital microbial growth inhibitors (Orak et al., 2011). Tannins in pomegranate had antibacterial activity against streptococci (Kunte et al., 2018), E. coli, bacilli, and S. aureus (Pagliarulo et al., 2016). The synthetic resin compounds in pomegranates possess inhibitory and atom scavenging activities (Dreher et al., 2017). The banana peel has been found to carry highest inhibitors and antimicrobial activities (Vu et al., 2018), and possessed antifungal activities (Velázquez-Nuñez et al., 2013). These findings were further verified by the results of Okeke et al. (2015) where the antibacterial activity of citrus fruit were studied in great details.
Pathogen Zone of inhibition in mm (mean ± SEM)
Cepharadin Ceftriaxone Pipemidic acid Ofloxacin
Bacillus subtilis 1.66 ± 0.27 1.33 ± 0.27 7.60 ± 0.27 12.66 ± 0.27
Micrococcus luteus 1.33 ± 0.33 1.00 ± 0.00 7.66 ± 0.33 12.66 ± 0.33
Pseudomonas aeruginosa 1.66 ± 0.33 1.66 ± 0.33 14.33 ± 0.33 8.33 ± 3.17
Staphylococcus aureus 1.00 ± 0.00 1.00 ± 0.00 1.33 ± 0.27 0.00
Klebsiella pneumonia 1.66 ± 0.33 1.00 ± 0.00 8.60 ± 0.33 14.00 ± 0.57
Escherichia coli 1.33 ± 0.33 1.66 ± 0.33 14.00 ± 0.57 13.00 ± 0.57
Table 2. Antibiogram analysis against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, and Staphylococcus aureus.
Figure 1. Antibiogram analysis against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, and Staphylococcus aureus.Note: a = Bacillus subtilis; b = Klebsiella pneumonia; c = Escherichia coli; d = Micrococcus luteus; e = Staphylococcus aureus; f = Pseudomonas aeruginosa; CPD = cepharadin; CTX = ceftriaxone; PMA = pipemidic acid; OFX = ofloxacin.
Figure 2. Antibiogram analysis of antibiotics and fruit peels against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Micrococcus luteus, Klebsiella pneumoniae, and Staphylococcus aureus. Each bar represents the mean value of three replicates (n = 3) ± SEM; * = the significant difference between antibiotics and fruit peels; *** = p ≤ 0.001.
Gillani, S. F., et al./IFRJ 27(5) : 963 - 970968
Conclusion
The preliminary phytochemical analysis demonstrated in the present work highlights the potential of fruit peels in carrying multiple and crucial bioactive compounds. This potential may used for the development of novel therapeutics in the future. Assessment of the antibacterial activity of fruit peels revealed a significant zone of inhibition against important human pathogens. The results obtained also confirmed that the studied microorgan-isms were more susceptible to fruit peels than stand-ard antibiotics, highlighting the capabilities of fruit
peels to substitute synthetic drugs and leveraging the development of potent novel antibiotics.
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